Cardiovascular disease in the United States is the leading cause of death (AHA). The long term objective of this research study is to understand basic cholesterol metabolism in order to prevent and combat diseases of cholesterol dysfunction such as atherosclerosis (heart attack, stroke), Newman Pick-C and Alzheimer's. The ability of cells to control cholesterol levels is a balance between the de novo synthesis and uptake of cholesterol versus secretion and efflux of cholesterol. Excess cholesterol in cells is toxic and is neutralized by esterification and stored as cholesteryl esters (CE). Hydrolysis of CE is necessary for the utilization of cholesterol for bile synthesis, hormone production, efflux of excess cholesterol and many other cellular functions. Our hypothesis is that, ES-4 is a hepatic neutral cholesteryl ester hydrolase (NCEH), and propose the following experiments. In our first aim we seek to define and understand the contribution of ES- 4 in CE hydrolysis. ES-4 protein will be suppressed or overexpressed using siRNA or transfection strategies. Using hepatic cells with various ES-4 levels, CE turnover, cholesterol mass, selective uptake and direct measurements of CE hydrolysis will be determined. The second aim of the study tests the hypothesis that the 5-kb region upstream of ES-4 transcriptional start site contains regulatory regions and these regions are necessary for the induction elicited by cholesterol and LXR agonist. This hypothesis will be tested using cholesterol loading/unloading, treatments with LXR agonist in conjunction with reporter constructs. Identifying the hepatic NCEH has significant basic science and clinical implications;it provides a new target for reducing VLDL/LDL CE production and would provide further understanding of the hepatic cholesterol cycle. Atherosclerosis (hardening of arteries due to excess cholesterol) mediated diseases (heart attack and strokes) are the leading cause of illness and death in the United States (NHLBI). This proposal examines ES-4, potentially a key player in modulating cholesterol levels/metabolism in the liver. Research in cholesterol metabolism is vital when considering that decreasing total cholesterol levels by 10% may result in an estimated 30 percent reduction in the incidence of coronary heart disease (CDC-2000).